Commentary Mining copper transport genes Nancy C. Andrews* Howard Hughes Medical Institute, Department of Medicine, Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115 opper is not only a ubiquitous metal in Transfection studies showed that the known copper-containing enzymes have Cour modern, technological environ- mammalian homologs also could stimu- highly specialized functions, and none is ment, it is also essential for the function of late copper accumulation in animal cells an obvious candidate for the cause of most living organisms. Just as it allows for (2, 9). Although their normal subcellular embryonic lethality, although Kuo et al. the movement of electrons through wires, localization has not been described, the (1) have speculated that lysyl oxidase it helps catalyze the movement of elec- transfection results suggest that they are might be important for formation of em- trons within biological molecules. Making expressed on the cell surface. bryonic structures. up only 0.01% of the Earth’s crust, it is These data indicated that mammalian There are at least two alternative hy- relatively scarce in the environment and CTR1 proteins could act as copper trans- potheses that should be entertained. First, must be actively scavenged. Until recently, porters, but did not prove that they serve both groups modified the murine Ctr1 little was known about how trace amounts that function in vivo in their normal con- locus by replacement of the gene with a of dietary copper are assimilated by intes- text. To investigate that question, two new transcriptional unit. It is plausible tinal absorptive cells to enter the body. In laboratories (1, 2) undertook targeted that the embryonic lethality is not a result this issue of PNAS, three groups report gene disruption of murine Ctr1. The re- of loss of Ctr1, but rather the result of genetic studies that may give insight into sults of the two knockout experiments are altered expression of another nearby gene. this process. Kuo et al. (1) and Lee et al. similar, but not quite identical. Lee et al. (2) mention that they have (2) report targeted gene disruption of a Both groups replaced all four exons of rescued the knockout animals through putative copper uptake molecule, Ctr1. In the compact Ctr1 gene with a neomycin transgenic expression of Ctr1, but details an accompanying report, Hamza and col- selection cassette. They transmitted the are not provided. The possibility that leagues (3) report disruption of the gene disrupted allele through the mouse germ there is alteration of expression of a crit- encoding a copper chaperone, which es- line and analyzed heterozygous mice for ical nearby gene could be eliminated by corts copper to sites of use and export copper content. Intriguingly, tissue cop- retargeting of the Ctr1 locus with an ex- within the cell. per levels were about half normal in cisable selection marker. However, the The story begins in yeast. For copper, spleen (2) and brain (1, 2), consistent with expression pattern of the Xenopus ho- COMMENTARY more than any other essential metal, Sac- a role for Ctr1 either in intestinal uptake molog, Xem1, argues that that experiment charomyces cerevisiae has proven to be a of copper or entry of copper into those is probably unnecessary (10). model model system. Ironically, insights tissues. The heterozygotes were otherwise Alternatively, although murine Ctr1 into copper metabolism initially came indistinguishable from certainly can act as a from experiments aimed at understanding wild-type mice. In con- copper transporter, how cells take up iron. Dancis, Kaplan, trast, the phenotype of it remains possible Klausner, and colleagues (4, 5) set up homozygous Ctr1Ϫ͞Ϫ Ironically, insights into that it also transports genetic screens to find mutations that mice was dramatic. copper metabolism another metal, such protected yeast cells from excess environ- They invariably died in as nickel or zinc, mental iron, hoping to identify compo- utero, but the timing of initially came from both neighbors to nents of the high-affinity iron uptake sys- embryonic demise dif- experiments aimed at copper in the peri- fered slightly between odic table. This is not tem. One of the first genes they found understanding how cells encoded a putative transmembrane trans- the two experiments. far-fetched—zinc is port protein that, to their surprise, had no Kuo et al. (1) found no take up iron. known to compete affinity for iron, but rather transported remnants of Ctr1Ϫ͞Ϫ with copper for in- copper. This protein, designated Ctr1p, embryos after embry- testinal absorption. supplies the metal for a multicopper fer- onic day 9.5 (E9.5). Severe developmental Furthermore, the mammalian iron trans- roxidase needed for high affinity iron defects were detectable as early as E6.5. In porter DMT1 (also known as Nramp2, Ϫ͞Ϫ transport. contrast, Lee et al. (2) found Ctr1 DCT1) and the Arabidopsis iron trans- Until that time, little was known about embryos in Mendelian proportions as late porter IRT1 both have been shown to be Ϫ͞Ϫ how copper enters eukaryotic cells, al- as E10.5. In both cases, Ctr1 embryos promiscuous in their transport of heavy though some details of copper export had were highly abnormal, with severe growth metal ions (11–13). It is unclear whether been elucidated through studies of human retardation dating to E6.5–E7.5, impaired mammals require nickel for cellular me- patients with Menkes disease and Wilson gastrulation, developmental defects in tabolism, but they certainly require zinc, disease (to be discussed later). But inves- both neural ectodermal and mesodermal and relatively little is known about how tigators were quick to exploit the yeast cell layers, and diminished mesenchymal zinc enters cells. It might be, for example, data. Using Ctr1p-deficient yeast cells as cell formation and͞or migration. living test tubes, plant (6), human (7), and These results indicate that Ctr1 plays an that zinc is required at an early develop- mouse (8) cDNAs were isolated that res- essential role early in embryogenesis. This cued their copper-deficient phenotype. is surprising if one interprets the data to Gratifyingly, in each case the cDNAs en- conclude that copper is limiting for a very See companion articles on pages 6836, 6842, and 6848. coded proteins with homology to Ctr1p. early function. As discussed below, the *E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.131192498 PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6543–6545 Downloaded by guest on September 30, 2021 mental time when Ctr1 is the only uptake However, biochemical studies using fibro- (designated ATP7B) encodes a P-type system available. blasts from two affected family members ATPase, WND, that is highly expressed in If the early lethality is due to copper suggested that cellular copper accumula- the liver (28–30). WND functions within deficiency, it is reasonable to conclude tion is increased, rather than decreased as the secretory apparatus, loading cytoplas- that Ctr1 is the exclusive transporter re- was expected (19). mic copper onto the plasma ferroxidase sponsible for mammalian copper uptake, An alternative candidate for the defect ceruloplasmin, which facilitates iron ex- at least early in embryogenesis. This would responsible for familial hypocupremia port from storage cells. be an important distinction between uni- (18) might be proposed based on a report Once again, there is a spontaneous cellular and multicellular eukaryotes, be- from Hamza and colleagues (3), also in mouse mutant with features similar to the cause yeast has redundancy in its copper this issue of PNAS, describing targeted human disease. Toxic milk (tx) mice were uptake genes. In addition to CTR1, S. disruption of the murine Atox1 gene. first identified because the offspring of cerevisiae has a second high-affinity cop- However, to understand the function of homozygous mutant mothers demon- per transporter gene, CTR3 (which is in- Atox1, it is necessary to consider not strated poor growth, experienced hypo- terrupted by a transposon in many lab copper uptake, but copper egress from pigmentation and tremors, and died be- strains; ref. 14), and a low-affinity trans- cells. fore weaning (32). This was shown to be porter gene, CTR2. In humans (and prob- Two copper export proteins initially caused by a lack of copper in the mother’s ably also in mice), a CTR-related homolog were discovered through identification of milk. Homozygous animals initially ap- is located close to CTR1 in the genome, the genes responsible for human diseases peared normal, but on closer study they but its function remains uncertain (7). with very different phenotypes. Menkes were found to have increased concentra- Obviously, neither the mammalian CTR disease has features of a severe copper tions of copper in the liver and decreased homolog nor DMT1, which is capable of deficiency disorder with X-linked reces- ceruloplasmin and copper levels in the copper transport in an oocyte assay (11), sive inheritance (20). Affected infants typ- plasma. These abnormalities all result can substitute for Ctr1 activity in the ically have growth retardation, severe from mutations in the murine Atp7b or- Ctr1Ϫ͞Ϫ mice. neurological impairment, mental retarda- tholog. The gene also has been disrupted The severity of the Ctr1Ϫ͞Ϫ phenotype tion, seizures, and abnormal hair and bone by deliberate gene targeting, to produce a is striking when compared with that of the abnormalities, leading to death before 5 mouse model of Wilson disease that lacks microcytic anemia (mk) mouse, which car- years of age. There is a milder variant, WND altogether (33). ries a severe loss-of-function mutation in designated X-linked cutis laxa or occipital S. cerevisiae also has a copper transport- the iron transporter DMT1 (15). Like horn syndrome, which is not lethal (re- ing P-type ATPase, designated Ccc2p.